Field of the Invention
The present invention provides methods for making heteromultimeric molecules, such as bispecific antibodies, and compositions comprising said molecules. The methods include introducing substitutions in amino acids that are in contact at the interface between two polypeptides. Such substitutions include changes in amino acids that result in altered electrostatic and/or hydrophobic/hydrophilic interactions between the polypeptides that make up the heteromultimeric molecules. The substitutions result in multimeric molecules in which homomultimeric molecules are disfavored, and heteromultimeric molecules are preferentially formed.
Background Art
The hallmark of monoclonal antibodies is their ability to specifically bind to a particular antigen, which enables them to bind to their target in vivo but not antigen-negative sites. Once bound to a target, therapeutic monoclonal antibodies can deliver a toxic payload, act as agonists or antagonists of receptors, or as neutralizers of ligands. Monoclonal antibodies can also be modified to be more immunologically tolerated in various species. One such modification, that entails the replacement of amino acids in structural regions with amino acids found in humans, is humanization. Humanized antibodies can then be further modified. One such modification is arming the humanized antibody with additional cytotoxic mechanisms, be it radioisotopes, bacterial toxins, inflammatory cytokines, chemotherapeutics or prodrugs.
There is a growing number of approved cancer therapeutics that are efficacious either as a chimerized antibody (Rituximab) or humanized IgG1 (Herceptin and Campath-1H), as conjugate with chemotherapeutics (Mylotarg) or a radioisotope (Zevalin and Bexxar). In spite of this progress, the efficacy of monoclonal antibodies for cancer treatment is still limited, leaving great potential for further improvements. One class of antibody derivatives with the promise of enhanced potency for cancer treatment is bispecific antibodies.
Antibodies with a dual specificity in their binding arms usually do not occur in nature and, therefore, were developed through recombinant DNA or cell-fusion technology. Most bispecific antibodies are designed to recruit cytotoxic effector cells of the immune system effectively against pathogenic target cells. After more than 15 years of extensive research, many different types of bispecific antibodies have been developed but only a few have advanced to clinical trials.
Among the first bispecific antibodies were constructs designed to redirect T cells against cancer target cells. Target cells are killed when cytotoxic T lymphocytes (CTLs) are tethered to tumor cells and simultaneously triggered by one arm of the bispecific antibody that interact with the T-cell receptor (TCR)-CD3 complex. CTLs, which are considered to be the most potent killer cells of the immune system, cannot be engaged by monoclonal antibodies because they lack Fcγ-receptors.
Another type of bispecific antibody is those that simultaneously bind tumor cells and an activating Fcγ-receptor, for example, CD64/FcγRI on monocytes. Binding of this type of bispecific antibody to Fcγ-receptors can elicit effector cell activation, without being competed by simultaneously binding normal IgG.
One method for generating bispecific antibodies has been termed the “knobs-into-holes” strategy (see, e.g., WO 2006/028936). The mispairing of Ig heavy chains is reduced in this technology by mutating selected amino acids forming the interface of the CH3 domains in human IgG. At positions within the CH3 domain at which the two heavy chains interact directly, an amino acid with a small side chain (hole) is introduced into the sequence of one heavy chain and an amino acid with a large side chain (knob) into that of the other one. As a result, the protein interaction between knobs and holes has been described as leading to the formation of up to 90% of the correct bispecific human IgG by transfected mammalian host cells.
The present invention provides methods for preferentially forming heteromultimeric molecules by mutating selected amino acids that interact at the interface between two polypeptides by replacing an amino acid residue involved in hydrophilic interactions with a more hydrophobic amino acid residue and/or replacing an amino acid involved in a charge interaction with another amino acid.